Prostate cancer is characterized by dependence on androgens for growth and drugs that target the androgen receptor has been the mainstay of therapy. Under selection pressure of treatment, prostate cancer evolves resistance mechanisms to grow despite therapy. In a subset of patients, the loss of prostate lineage specification results in resistance to all therapy directed at the androgen receptor and there are few therapeutic options. We have recently identified two mechanism of lineage plasticity prostate cancer that are reversible by epigenetic therapy. The overall goal is to develop combination therapy to reverse or prevent lineage plasticity and prolong response. First, we have observed that tumors with combined TP53 and RB1 loss progress to androgen receptor independent disease that required activity of EZH2. This suggests that EZH2 is a therapeutic target for this subset of prostate cancer. Second, we discovered that a subset of prostate cancer aberrantly express HNF4G and HNF1A, master regulators of the gastrointestinal lineage, and bypass the prostate lineage requirement for androgen signaling. Inhibition with BET inhibitors inhibit this pathway and re- sensitize prostate cancer to androgen receptor directed therapy. In Aim 1, we will test the in vivo activity of EZH2 inhibition in AR-negative prostate cancer in combination with the antiandrogen enzalutamide. In Aim 2, we will test the in vivo activity of BET inhibition in HNF4G/HNF1A positive prostate cancer in combination with enzalutamide. In Aim 3, we will perform preclinical studies of epigenetic inhibitor of BET, EZH2, and P300/CBP in a large panel of patient-derived models of prostate cancer that reflect the clinical heterogeneity of human prostate cancer. For our studies, we will use clinically relevant drugs that are either in clinical trials or are first in class with clinical potential. The proposed research is innovative because it tests a novel paradigm that loss of prostate lineage is reversible and druggable, and is significant because of the potential to rapidly develop novel treatment to patients with few options.